DESCRIPTION: (Verbatim from the Applicant's Abstract) While caspase-3 has been widely implicated in apoptosis, rapid progress in understanding mechanisms of apoptotic cell death is difficult in in vivo models of traumatic brain injury (TBI). Traumatic brain injury is multifactorial, primarily including tissue deformation and secondary hypoxia and ischemia, all of which could result in release of excitotoxic levels of glutamate. We have developed a battery of in vitro systems to study separately three major pathological components of traumatic brain injury: (1) biomechanical stretch injury, (2) ischemia and/or hypoxia (oxygen and/or glucose deprivation)and (3) glutamate-induced excitotoxicity. Aim 1 will characterize caspase-3 and calpain activation in these in vitro models and examine the contribution of these two families of cysteine proteases to archetypal apoptotic and necrotic cell death phenotypes. Researchers have described at least two major apoptotic cell death pathways in mammals regulating cspase-3 activation: (1) a mitochondrial pathway requiring cspase-9 activation and (2) a receptor mediated signal transduction pathway principally involving tumor necrosis factor (TNF-a) and requiring caspase-8/10 activation. Both of these pathways could regulate caspase-3 activation. AIM 2 will examine the mitochondrial cell death pathway since this pathway is well characterized in non- neuronal systems, the requisite cellular machinery is present in neurons and data implicate a mitochondrial contribution to neuronal injury and apoptotic cell death. Aim 3 will examine TNF-a receptor coupled pathway since laboratory and clinical studies suggest that traumatic brain injury increases levels of TNF-a that could contribute to apoptotic neuronal death. Aim 4 will test the generality of in vitro mechanisms of cell death characterized in Aims 1-3 in a clinically relevant in vivo model of traumatic brain injury. Our general hypothesis is that caspase-3 activation by casepase-9 and/or caspase 8/10 is an important common mediator of apoptotic cell death following CNS injury. Calpain activation contributes primarily to necrosis and, to a lesser extent, apoptosis.